Table 1.
Primer sequences for Homo sapiens.
Table 2.
Primer sequences for Mus musculus.
Fig 1.
FdU depletion of non neuronal cells.
(A) Control culture of mouse embryonic cortical neurons stained for neurons (Map2, green), astrocytes (GFAP, red) and DAPI (blue). (B) Sister culture of A treated with 5 μM FdU shows reduced astrocyte presence but also damaged neurons. (C-D) Low concentration FdU treatment (C) Control, (D) 1 μM, (E) 2 μM FdU applied at DIV 4 for 24hr followed by growth until DIV 15. (F-M) No evidence for quiescent non-neuronal cells. Control (F,H,J,L) or FdU treated (G,I,K,M) cortical cultures were stimulated with LPS (H,I,L,M) then stained for the GFAP (F-I) or NG2 (J-M). The white arrows in Panel L illustrate NG2-positive astrocyte-like cells that appear in LPS-treated control cultures. Scale bar = 50μm. *: p< 0.05. n = 4 for mixed culture and n = 3 for FdU culture. (N) Western blot of GFAP levels in normal (Lanes 1,2) or FdU-treated (Lanes 3,4) cultures after LPS treatment. Actin served as a loading control. (O) Western blots of GFAP or NG2 in control (Lanes 1–4) and FdU treated (Lanes 5–7) cortical neuron cultures. (P) Quantification of the levels of GFAP (normalized to the levels of actin). (Q) Quantification of the normalized levels of NGS.
Fig 2.
FdU killed proliferating cells while maintained neuronal survival.
Cell cycle activity in neurons (Map2-positive cells) was monitored either with EdU (A,C) or Ki67 (B,D) in control (A,B) or FdU-treated (C,D) cells. (E-H) Quantification of the number of double labeled cells. Scale bar = 50μm. *: p< 0.05, **: p< 0.01. n = 4 for mixed culture and n = 3 for FdU culture.
Fig 3.
FdU treatment does not significantly diminish synaptic density.
Synaptic structures in mixed (A,B) or FdU-treated (C,D) cultures were immunostained with pre-synaptic (synapsin-I, A,C) or postsynaptic (Homer-1, B,D) markers. Insets illustrate individual neurons stained with the indicated synaptic markers. Scale bar = 50 μm. n = 4 for mixed cultures and n = 3 for FdU cultures. (E-G) Protein expression was monitored by Western blot of different pre- (synapsin-I) and post- (Homer1) synaptic markers. (F,H) Quantification of the Western blots normalized to the actin loading control.
Fig 4.
L-glutamate excitotoxicity is retained in the FdU culture.
Both mixed (A, B) and FdU-treated (C, D) cultures showed loss of MAP2 positive cells after 24hr L-glutamate treatment. (E) Quantification confirmed the qualitative results. Scale bar = 50μm. ***: p< 0.001. n = 3 for each group.
Fig 5.
Differential expression of NFκB subunits and cytokine receptor genes in mixed and FdU-treated neuronal cultures.
(A) Western blot of various NFκB subunits as indicated in control (Lanes 1,2) and FdU-treated (Lanes 3.4). TNFα treatment (Lanes 2,4) was used to measure the inflammatory response of the cultured cells. See text for details. (B) Quantification of the Western results shown in Panel A. (C-H) Immunostaining for p50 (C,D,G) and p65 (E,F,H) in FdU-treated (D,F) and untreated (C,E) cultures. (G-H) The NFκB antagonist, celastrol, confirmed the specificity of NFκB signals. Scale bar = 50μm. *: p< 0.05, **: p< 0.01, ***: p< 0.001. n = 3 for each group. (I) Q-PCR values of transcripts of various cytokine receptors (X-axis) in FdU-treated cultures are shown as ratios of the values found in untreated cultures.
Fig 6.
Inflammation induced neuronal damage requires the presence of non-neuronal cells.
Addition of 12.5% THP-1 amyloid conditional medium (AM) triggered both neuronal death and cell cycle reentry in mixed culture (A-B) but failed to do so in FdU-treated culture (C-D). Quantification of Map2 cell numbers (E) and neuronal cell cycle activity (F) confirmed these qualitative observations. (G) AM induced cytokine gene expression in mixed culture as measured by qPCR (all values relative to THP-1 control). Application of individual pro-inflammatory cytokines validated the findings with AM treatment, both with respect to neuronal cell numbers (H) and cell cycle events (I). Scale bar = 50 μm. *: p < 0.05, **: p < 0.01, ***: p < 0.001. n = 3 for each group.